Heterodimeric proteins with fc mutations

Abstract

Provided are heterodimeric proteins comprising polypeptides having CH3 domains with engineered residues that form disulfide bonds and/or salt bridges. Also provided are activatable antibodies targeting CD3 and/or HER2. Compositions, methods of manufacture and methods of treatment using the heterodimeric proteins and the activatable antibodies are further provided.

What is claimed is: 
     
         1 . A heterodimeric protein comprising a first polypeptide comprising a first immunoglobulin heavy chain constant domain 3 (CH3 domain) and a second polypeptide comprising a second CH3 domain, wherein:
 i) the first CH3 domain comprises a cysteine (C) residue at position 390 and the second CH3 domain comprises a cysteine residue at position 400, or the first CH3 domain comprises a cysteine residue at position 400 and the second CH3 domain comprises a cysteine residue at position 390; or   ii) the first CH3 domain comprises a cysteine residue at position 392 and the second CH3 domain comprises a cysteine residue at position 397, or the first CH3 domain comprises a cysteine residue at position 397 and the second CH3 domain comprises a cysteine residue at position 392; or   iii) the first CH3 domain comprises a cysteine residue at position 392 and the second CH3 domain comprises a cysteine residue at position 400, or the first CH3 domain comprises a cysteine residue at position 400 and the second CH3 domain comprises a cysteine residue at position 392; and   wherein the amino acid residue numbering is based on EU numbering.   
     
     
         2 . The heterodimeric protein of  claim 1 , wherein:
 i) the first CH3 domain comprises N390C substitution and the second CH3 domain comprises S400C substitution, or the first CH3 domain comprises S400C substitution and the second CH3 domain comprises N390C substitution; or   ii) the first CH3 domain comprises K392C substitution and the second CH3 domain comprises V397C substitution, or the first CH3 domain comprises V397C substitution and the second CH3 domain comprises K392C substitution; or   iii) the first CH3 domain comprises K392C substitution and the second CH3 domain comprises S400C substitution, or the first CH3 domain comprises S400C substitution and the second CH3 domain comprises K392C substitution.   
     
     
         3 . The heterodimeric protein of  claim 1  or  claim 2 , wherein:
 i) the first CH3 domain further comprises a positively charged residue at position 357 and the second CH3 domain further comprises a negatively charged residue at position 351, or the first CH3 domain further comprises a negatively charged residue at position 351 and the second CH3 domain further comprises a positively charged residue at position 357; or 
 ii) the first CH3 domain further comprises a positively charged residue at position 411 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain further comprises a negatively charged residue at position 370 and the second CH3 domain further comprises a positively charged residue at position 411; or 
 iii) the first CH3 domain further comprises a positively charged residue at position 364 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain further comprises a negatively charged residue at position 370 and the second CH3 domain further comprises a positively charged residue at position 364; or 
 a combination of i) and ii), or a combination of i) and iii); and 
 wherein the amino acid residue numbering is based on EU numbering. 
 
     
     
         4 . The heterodimeric protein of any one of  claims 1 - 3 , wherein the first CH3 domain further comprises a positively charged residue at position 356 and the second CH3 domain further comprises a negatively charged residue at position 439, or first CH3 domain further comprises a negatively charged residue at position 439 and the second CH3 domain further comprises a positively charged residue at position 356; and wherein the amino acid residue numbering is based on EU numbering. 
     
     
         5 . The heterodimeric protein of  claim 3  or  claim 4 , wherein:
 i) the positively charged residue is a lysine (K) residue, and the negatively charged residue is an aspartic acid (D) residue; or 
 ii) the positively charged residue is a lysine (K) residue, and the negatively charged residue is a glutamic acid (E) residue; or 
 iii) the positively charged residue is an arginine (R) residue, and the negatively charged residue is an aspartic acid (D) residue; or 
 iv) the positively charged residue is an arginine (R) residue, and the negatively charged residue is a glutamic acid (E) residue. 
 
     
     
         6 . The heterodimeric protein of  claim 5 , wherein:
 i) the first CH3 domain comprises E357K and T411K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and T411K substitutions; or   ii) the first CH3 domain comprises E357K and S364K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and S364K substitutions; or   iii) the first CH3 domain comprises D356K, E357K and S364K substitutions and the second CH3 domain comprises L351D, K370D and K439D substitutions, or the first CH3 domain comprises L351 D, K370D and K439D substitutions and the second CH3 domain comprises D356K, E357K and S364K substitutions.   
     
     
         7 . The heterodimeric protein of any one of  claims 1 - 5 , wherein:
 i) the first CH3 domain further comprises K392D and K409D substitutions and the second CH3 domain further comprises D356K and D399K substitutions, or the first CH3 domain further comprises D356K and D399K substitutions and the second CH3 domain further comprises K392D and K409D substitutions; or   ii) the first CH3 domain further comprises L368D and K370S substitutions and the second CH3 domain further comprises E357Q and S364K substitutions, or the first CH3 domain further comprises E357Q and S364K substitutions and the second CH3 domain further comprises L368D and K370S substitutions; or   iii) the first CH3 domain further comprises L351K and T366K substitutions and the second CH3 domain further comprises L351D and L368E substitutions, or the first CH3 domain further comprises L351D and L368E substitutions and the second CH3 domain further comprises L351K and T366K substitutions; or   (iv) the first CH3 domain further comprises P395K, P396K and V397K substitutions and the second CH3 domain comprises T394D, P395D and P396D substitutions, or the first CH3 domain further comprises T394D, P395D and P3% D substitutions and the second CH3 domain further comprises P395K, P396K and V397K substitutions; or   (v) the first CH3 domain further comprises F405E, Y407E and K409E substitutions and the second CH3 domain comprises F405K and Y407K substitutions, or the first CH3 domain further comprises F405K and Y407K substitutions and the second CH3 domain further comprises F405E, Y407E and K409E substitutions.   
     
     
         8 . The heterodimeric protein of  claim 6 , wherein
 i) the first CH3 domain comprises E357K, S364K and N390C substitutions and the second CH3 domain comprises L351D, K370D, and S400C substitutions, or the first CH3 domain comprises L351D, K370D, and S400C substitutions and the second CH3 domain comprises E357K, S364K and N390C substitutions; or   ii) the first CH3 domain comprises E357K, S364K and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K and S400C substitutions; or   iii) the first CH3 domain comprises D356K, E357K, S364K and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K and S400C substitutions; or   iv) the first CH3 domain comprises D356K, E357K, S364K and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D and S400C substitutions, or the first CH3 domain comprises L351D, K370D, K439D and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K and N390C substitutions.   
     
     
         9 . The heterodimeric protein of any one of  claims 1 - 8 , wherein the first CH3 domain and the second CH3 domain further comprise knob-into-hole residues. 
     
     
         10 . The heterodimeric protein of  claim 9 , wherein:
 i) the first CH3 domain comprises T336S, L368A and Y407V substitutions and the second CH3 domain comprises T366W substitution, or the first CH3 domain comprises T366W substitution and the second CH3 domain comprises T336S, L368A and Y407V substitutions; or   ii) the first CH3 domain comprises L368V and Y407V substitutions and the second CH3 domain comprises T366W substitution, or the first CH3 domain comprises T366W substitution and the second CH3 domain comprises L368V and Y407V substitutions.   
     
     
         11 . A heterodimeric protein comprising a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein:
 i) the first CH3 domain comprises a positively charged residue at position 357 and the second CH3 domain comprises a negatively charged residue at position 351, or the first CH3 domain comprises a negatively charged residue at position 351 and the second CH3 domain comprises a positively charged residue at position 357; or   ii) the first CH3 domain comprises a positively charged residue at position 411 and the second CH3 domain comprises a negatively charged residue at position 370, or the first CH3 domain comprises a negatively charged residue at position 370 and the second CH3 domain comprises a positively charged residue at position 411; or   iii) the first CH3 domain comprises a positively charged residue at position 364 and the second CH3 domain comprises a negatively charged residue at position 370, or the first CH3 domain comprises a negatively charged residue at position 370 and the second CH3 domain comprises a positively charged residue at position 364; and wherein the amino acid residue numbering is based on EU numbering.   
     
     
         12 . The heterodimeric protein of  claim 11 , wherein the first CH3 domain comprises a charged residue at position 356 and the second CH3 domain comprises a negatively charged residue at position 439, or first CH3 domain comprises a negatively charged residue at position 439 and the second CH3 domain comprises a positively charged residue at position 356; and wherein the amino acid residue numbering is based on EU numbering. 
     
     
         13 . The heterodimeric protein of  claim 11  or  claim 12 , wherein:
 i) the positively charged residue is a lysine (K) residue, and the negatively charged residue is an aspartic acid (D) residue; or 
 ii) the positively charged residue is a lysine (K) residue, and the negatively charged residue is a glutamic acid (E) residue; or 
 iii) the positively charged residue is an arginine (R) residue, and the negatively charged residue is an aspartic acid (D) residue; or 
 iv) the positively charged residue is an arginine (R) residue, and the negatively charged residue is a glutamic acid (E) residue. 
 
     
     
         14 . The heterodimeric protein of  claim 13 , wherein:
 i) the first CH3 domain comprises E357K and T411K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and T411K substitutions; or   ii) the first CH3 domain comprises E357K and S364K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and S364K substitutions; or   iii) the first CH3 domain comprises D356K, E357K and S364K substitutions and the second CH3 domain comprises L351D, K370D and K439D substitutions, or the first CH3 domain comprises L351 D, K370D and K439D substitutions and the second CH3 domain comprises D356K, E357K and S364K substitutions.   
     
     
         15 . The heterodimeric protein of any one of  claims 11 - 14 , wherein:
 i) the first CH3 domain further comprises K392C substitution and the second CH3 domain further comprises D399C substitution, or the first CH3 domain further comprises D399C substitution and the second CH3 domain further comprises K392C substitution; or   ii) the first CH3 domain further comprises Y394C substitution and the second CH3 domain further comprises S354C substitution, or the first CH3 domain further comprises S354C substitution and the second CH3 domain further comprises Y394C substitution; or   iii) the first CH3 domain further comprises D356C substitution and the second CH3 domain further comprises Y349C substitution, or the first CH3 domain further comprises Y349C substitution and the second CH3 domain further comprises D356C substitution.   
     
     
         16 . The heterodimeric protein of any one of  claims 1 - 15 , wherein the first CH3 domain and the second CH3 domain are human CH3 domains. 
     
     
         17 . The heterodimeric protein of any one of  claims 1 - 16 , wherein the first polypeptide and the second polypeptide each comprises from the N-terminus to the C-terminus at least a portion of an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2 domain), and the CH3 domain. 
     
     
         18 . The heterodimeric protein of  claim 17 , wherein the CH2 domains and the CH3 domains form an IgG Fc region. 
     
     
         19 . The heterodimeric protein of  claim 18 , wherein the Fc region is of the human IgG1 subclass. 
     
     
         20 . The heterodimeric protein of  claim 18 , wherein the Fc region is of the human IgG4 subclass. 
     
     
         21 . The heterodimeric protein of  claim 20 , wherein the Fc region further comprises S228P substitution. 
     
     
         22 . The heterodimeric protein of any one of  claims 18 - 20 , wherein the Fc region further comprises N297A substitution. 
     
     
         23 . The heterodimeric protein of any one of  claims 1 - 22 , the first polypeptide and the second polypeptide are antibody heavy chains, and wherein the heterodimeric protein further comprises one or more antibody light chains. 
     
     
         24 . The heterodimeric protein of  claim 23 , wherein the heterodimeric protein is a multispecific antibody. 
     
     
         25 . The heterodimeric protein of  claim 24 , further comprising a third polypeptide and a fourth polypeptide, wherein:
 (i) the first polypeptide comprises a structure represented by the formula:
   VH1-CH1-hinge-CH2-first CH3-L1-scFv1  (Ia);
 
   (ii) the second polypeptide comprises a structure represented by the formula:
   VH2-CH1-hinge-CH2-second CH3-L2-scFv2  (IIa);
 
   (iii) the third polypeptide comprises a structure represented by the formula:
   VL1-CL  (Ib); and
 
   (iv) the fourth polypeptide comprises a structure represented by the formula:
   VL2-CL  (IIb);
 
   
       wherein:
 VL1 is a first immunoglobulin light chain variable domain; 
 VH1 is a first immunoglobulin heavy chain variable domain; 
 VL2 is a second immunoglobulin light chain variable domain; 
 VH2 is a second immunoglobulin heavy chain variable domain; 
 scFv1 is a first single-chain variable fragment; 
 scFv2 is a second single-chain variable fragment; 
 CL is an immunoglobulin light chain constant domain; 
 CH1 is an immunoglobulin heavy chain constant domain 1: 
 CH2 is an immunoglobulin heavy chain constant domain 2; 
 hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and
 L1 and L2 is each independently a bond or a peptide linker; 
 
 
       wherein VL1 and VH1 associate to form a first Fv that specifically binds to a first target; 
       wherein VL2 and VH2 associate to form a second Fv that specifically binds to a second target; 
       wherein scFv1 specifically binds to a third target; and 
       wherein scFv2 specifically binds to a fourth target. 
     
     
         26 . The heterodimeric protein of  claim 25 , wherein scFv1 and scFv2 are identical. 
     
     
         27 . The heterodimeric protein of  claim 25  or  26 , wherein VL1 and VL2 are identical. 
     
     
         28 . The heterodimeric protein of  claim 26  or  27 , wherein the first Fv specifically binds PDL1, the second Fv specifically binds CD137, and scFv1 and scFv2 specifically bind CTLA-4. 
     
     
         29 . The heterodimeric protein of  claim 24 , further comprising a third polypeptide, wherein:
 (i) the first polypeptide comprises a structure represented by the formula:
   VH-CH1-hinge-CH2-first CH3  (IIIa);
 
   (ii) the second polypeptide comprises a structure represented by the formula:
   scFv-hinge-CH2-second CH3  (IVa); and
 
   (iii) the third polypeptide comprises a structure represented by the formula:
   VL-CL  (IIIb);
 
   wherein:
 VL is an immunoglobulin light chain variable domain; 
 VH is an immunoglobulin heavy chain variable domain; 
 scFv is a single-chain variable fragment; 
 CL is an immunoglobulin light chain constant domain; 
 CH1 is an immunoglobulin heavy chain constant domain 1; 
 CH2 is an immunoglobulin heavy chain constant domain 2; 
 and hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; 
   wherein VL and VH associate to form an Fv that specifically binds to a first target; and   wherein the scFv specifically binds to a second target.   
     
     
         30 . The heterodimeric protein of  claim 29 , wherein the Fv specifically binds CD137 and the scFv specifically binds PDL1. 
     
     
         31 . The heterodimeric protein of  claim 24 , wherein the heterodimeric protein is an activatable antibody, wherein the heterodimeric protein comprises a third polypeptide, and wherein:
 (i) the first polypeptide comprises a structure represented by the formula:
   VH-CH1-hinge-CH2-first CH3  (Va);
 
   (ii) the second polypeptide comprises a structure represented by the formula:
   MM1-CM1-scFv-hinge-CH2-second CH3  (VIa); and
 
   (iii) the third polypeptide comprises a structure represented by the formula:
   MM2-CM2-VL-CL  (IVb);
 
   wherein:
 VL is an immunoglobulin light chain variable domain; 
 VH is an immunoglobulin heavy chain variable domain; 
 scFv is a single-chain variable fragment; 
 CL is an immunoglobulin light chain constant domain; 
 CH1 is an immunoglobulin heavy chain constant domain 1; 
 CH2 is an immunoglobulin heavy chain constant domain 2; 
 hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; 
 MM1 is a first masking peptide; 
 MM2 is a second masking peptide; 
 CM1 is a first cleavable peptide; and 
 CM2 is a second cleavable peptide; 
   wherein VL and VH associate to form a first Fv that specifically binds to a first target;   wherein the scFv specifically binds to a second target;   wherein MM1 inhibits the binding of the scFv to the first target when CM1 is not cleaved; and   wherein MM2 inhibits the binding of the first Fv to the second target when CM2 is not cleaved.   
     
     
         32 . The heterodimeric protein of  claim 29  or  31 , wherein the first target is a tumor antigen, and the second target is CD3. 
     
     
         33 . The heterodimeric protein of  claim 31  or  32 , wherein MM1 comprises the amino acid sequence of SEQ ID NO: 35. 
     
     
         34 . The heterodimeric protein of  claim 32  or  33 , wherein the first target is HER2. 
     
     
         35 . The heterodimeric protein of  claim 34 , wherein MM2 comprises the amino acid sequence of SEQ ID NO: 36. 
     
     
         36 . An activatable antibody comprising: a first polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM comprises the amino acid sequence of SEQ ID NO: 35; wherein the MM inhibits binding of the activatable antibody to human CD3 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; and wherein:
 a) the TBM comprises a VL and the activatable antibody further comprises a second polypeptide comprising a VH;   b) the TBM comprises a VH and the activatable antibody further comprises a second polypeptide comprising a VL:   c) the TBM comprises from the N-terminus to the C-terminus, a VL and a VH; or   d) the TBM comprise from the N-terminus to the C-terminus, a VH and a VL; and   wherein the activatable antibody binds to human CD3 via the VH and VL when the CM is cleaved.   
     
     
         37 . An activatable antibody comprising: a first polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM comprises the amino acid sequence of SEQ ID NO: 36; wherein the MM inhibits binding of the activatable antibody to human HER2 when the CM is not cleaved;
 wherein the CM comprises at least a first cleavage site; and wherein:   a) the TBM comprises a VL and the activatable antibody further comprises a second polypeptide comprising a VH;   b) the TBM comprises a VH and the activatable antibody further comprises a second polypeptide comprising a VL;   c) the TBM comprises from the N-terminus to the C-terminus, a VL and a VH; or   d) the TBM comprise from the N-terminus to the C-terminus, a VH and a VL; and   wherein the activatable antibody binds to human HER2 via the VH and VL when the CM is cleaved.   
     
     
         38 . The activatable antibody of  claim 36  or  37 , comprising a first polypeptide, a second polypeptide and a third polypeptide, wherein:
 (i) the first polypeptide comprises a structure represented by the formula:
   VH-CH1-hinge-CH2-first CH3  (Va);
 
 
 (ii) the second polypeptide comprises a structure represented by the formula:
   MM1-CM1-scFv-hinge-CH2-second CH3  (VIa); and
 
 
 (iii) the third polypeptide comprises a structure represented by the formula:
   MM2-CM2-VL-CL  (IVb);
 
 
 wherein:
 VL is an immunoglobulin light chain variable domain; 
 VH is an immunoglobulin heavy chain variable domain; 
 scFv is a single-chain variable fragment; 
 CL is an immunoglobulin light chain constant domain; 
 CH1 is an immunoglobulin heavy chain constant domain 1; 
 CH2 is an immunoglobulin heavy chain constant domain 2; 
 hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; 
 MM1 is a first masking peptide; 
 MM2 is a second masking peptide; 
 CM1 is a first cleavable peptide; and 
 CM2 is a second cleavable peptide; 
 
 wherein VL and VH associate to form a first Fv that specifically binds to a first target; 
 wherein the scFv specifically binds to a second target; and wherein MM is MM1 or MM2. 
 
     
     
         39 . The heterodimeric protein of any one of  claims 32 - 35  or the activatable antibody of  claim 36  or  38 , wherein the first Fv or the TBM comprises a VH comprising an CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and/or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and/or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66. 
     
     
         40 . The heterodimeric protein of  claim 34  or  35 , or the activatable antibody of any one of  claims 37 - 39 , wherein the scFv or the TBM comprises a VH comprising an CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and/or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and/or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74 
     
     
         41 . The heterodimeric protein of any one of  claims 31 - 35  or the activatable antibody of any one of  claims 36 - 40 , wherein the activatable antibody comprises an Fc region comprising a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises D356K, E357K, S364K and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K and S400C substitutions. 
     
     
         42 . One or more nucleic acid(s) encoding the heterodimeric protein of any one of  claims 1 - 35  and  39 - 41  or the activatable antibody of any one of  claims 36 - 41 . 
     
     
         43 . A vector comprising the one or more nucleic acid(s) of  claim 42 . 
     
     
         44 . A host cell comprising the one or more nucleic acid(s) of  claim 42  or the vector of  claim 43 . 
     
     
         45 . A method for preparing a heterodimeric protein or an activatable antibody, comprising:
 (a) culturing the host cell of  claim 44  under conditions that allow expression of the one or more nucleic acid(s) or vector; and   (b) recovering the heterodimeric protein or the activatable antibody from the host cell culture.   
     
     
         46 . A pharmaceutical composition comprising the heterodimeric protein of 1-35 and 39-41 or the activatable antibody of any one of  claims 36 - 41 , and a pharmaceutically acceptable carrier. 
     
     
         47 . A method for treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 46 . 
     
     
         48 . The method of  claim 47 , wherein the disease or condition is cancer. 
     
     
         49 . The method of  claim 48 , wherein the cancer is lung cancer. 
     
     
         50 . The method of  claim 48 , wherein the cancer is ovarian cancer.